Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes.

Under conditions of low intracellular [Mg2+] ([Mg2+]i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (ICa) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in ICa in the Mg2+-deficient medium. Thus, the involvement of phosphorylation process in the increase in ICa was not likely. Effective suppression of this enhancement of ICa was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC50) was estimated to be 4.0 microM at pMg 6. This GTP-induced suppression of ICa is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed ICa similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg2+ and GTP on ICa. GTP is therefore assumed to bind directly to the Ca2+ channel. Interaction of Mg2+ and GTP with the Ca2+ channel activated in the Mg2+-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg2+]. The IC50 for GTP suppression was estimated to be 5.7 microM at pMg 6 and 6.9 microM at pMg 5. The results suggest strongly that Mg2+ and GTP independently bind and control Ca2+ channels.